L-Alanine Assay Kit (ab83394)

Thank you for contacting us yesterday in regards to the Alanine Transaminase Activity Assay Kit (ab105134). I have now had a chance to have a look at this product to see if it would be suitable to use in your experiment to assess the transaminase inhibition properties of your compounds.

In short, the kit should be compatible with this but would need to be modified in order to get the results that you are looking for. The kits is intended to be used to measure the inherent alanine transaminase activity of biological samples. For example, measuring the activity from liver tissue samples. This is performed by using the provided substrates for the enzyme to work on and measuring the pyruvate generated. The enzyme comes from the biological sample tested.

You would therefore need to introduce the alanine transaminase enzyme into your samples to measure the inhibition caused by your compounds. This enzyme is provided as the ALT positive control. What you would need to do is set up the reaction with this included in each of your wells and add your inhibitors. You could then compare the inhibition by observing the difference of the ALT activity in the presence and absence of inhibitor.

As the kit is not really designed for this purpose it may require some optimisation and the quantity of ALT provided is not very high as it is only intended to be used as a single positive control. You may want to consider looking at the L-Alanine Assay Kit (ab83394) instead. This kit allows for the detection of alanine but works on the same principle as ab105134. It uses ALT to convert alanine to pyruvate which is then specifically detected.

Again, this kit is not specifically desigend for the experiment you are hoping to perform and therefore the protocol would need to be modified. In this kit, the alanine standard is provided but may again be limiting in the experiment you are hoping to perform.

What I am going to do is see if either the ALT positive control for ab105134 or the alanine standard from ab83394 may be available to purchase independently of the kits. This may help you as you would be able to run more tests with each kit. Once I have this information I will get back to you.

I hope this information has been of help. If you have any further questions in the meantime, please do not hesitate to let me know.

Thank you for contacting us. I have contacted the lab regarding this for you. The product does contain alanine aminotransferase (ALT). I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us. I have spoken with the lab regarding the homogenization and wait peroid for use with this kit. They usually use a hand held dounce homogenizer, but pipetting back and forth in Assay buffer (it contains detergent) along with sonication can work. To check for efficient lysis, look at the sample (2 ul) under a microscope and to see if have few, if any, intact cells. Futher wait periods are not needed in this assay. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

* Generally, the usual mistake is that the probe is still frozen DMSO. Please make sure to warm the probe for 1-2 min in a 37 °C bath, pipette up/down to ensure probe is fully melted, then repeat the standard curve. Please send us the data if problem persists and we will be pleased to look into it further. * Merci de nous avoir contactés. Je suis désolée d'apprendre que vous avez eu des difficultés à obtenir de bons résultats avec notre kit ab83394. Mon collègue avait contacté le laboratoire pour savoir si il y avait des problèmes avec ce kit ou avec ce lot spécifique. Le laboratoire nous avait répondu qu'il n'ont pas eu des problèmes avec ce kit et que normalement, si un chercheur a un problème avec ce kit, c'est parce que la "probe" n'est pas décongelée complètement. Donc est-ce que vous pourriez donc s'il vous plaît répéter la courbe standard en vérifiant la "probe" de manière suivante: de préchauffer la "probe" dans un bain maire à 37 °C pour 1 à 2 minutes, de la resuspendre en pipetant la solution pour s'assurer que la "probe" est complètement décongelée. Si ceci ne permets pas de résoudre le problème, est-ce que vous pourriez nous envoyer les données pour que nous pouvons les analyser? Merci beaucoup pour votre collaboration. Je me réjouis de votre réponse.

All of the kits: ab65337, ab65342, ab83390, ab83394 are suitable for cell and tissue culture supernatants. As well as many other biological fluids like urine, plasma and serum.

Thank you for your inquiry. We generally recommend to lyse cells in the kits own assay buffer, as it is formulated with components conducive for the optimum reaction to occur. I hope this information helps. Please contact me with any other questions.